Corporate R&D Headquarters, Canon Inc Tokyo, Ohta-ku, Japan.
BMC Neurosci. 2012 Aug 16;13:101. doi: 10.1186/1471-2202-13-101.
Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood-brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB.
We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7-8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs.
The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs.
We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.
将化合物递送到大脑和视网膜是开发治疗药物和成像剂的挑战。这一挑战源于化合物内化进入大脑和视网膜分别受到血脑屏障（BBB）和血视网膜屏障（BRB）的限制。需要简单可靠的体内检测方法来鉴定可以轻易穿透 BBB 和 BRB 的化合物。
我们开发了六个荧光吲哚啉衍生物（IDs），并通过活体荧光成像在斑马鱼中检测它们穿透 BBB 和 BRB 的能力。将这些荧光 IDs 通过胚胎孵化后 7-8 天的斑马鱼幼体浸泡在含 ID 的培养基中，或者通过心脏内注射的方式给予活体斑马鱼。我们还使用多药耐药蛋白（MRP）的选择性抑制剂 MK571 来检测 MRP 对 ID 穿透 BBB 和 BRB 的通透性的影响。
通过简单浸泡给予这些屏障的 ID 的通透性与通过心脏内注射给予的 ID 的通透性相当。因此，这一发现支持了通过简单浸泡给药评估 ID 穿透 BBB 和 BRB 的通透性的有效性。利用这种斑马鱼模型，我们证明了这些荧光 IDs 中亚甲基链的长度显著影响它们通过 MRP 穿透 BBB 和 BRB 的能力。
我们证明了使用斑马鱼可以简单可靠地进行活体评估 BBB 和 BRB 对荧光 IDs 的通透性。荧光 IDs 的结构可以灵活修饰，因此可以使用这种基于斑马鱼的检测方法评估大量 ID 对 BBB 和 BRB 的通透性。获得的大量数据可能有助于进行计算机分析，以阐明化学结构与 BBB 和 BRB 中的外排转运蛋白之间相互作用的精确机制。反过来，理解这些机制可能会导致针对大脑和视网膜的化合物的高效设计。
